TGF-[unreadable] plays a complex but critical role in bone. TGF-[unreadable] inhibition of terminal osteoblast differentiation can result from its activation of the intracellular effector, SmadS, to bind and inhibit the function of Runx2, a critical transcriptional regulator of osteoblast differentiation. Preliminary data suggest that HDACs 4 and 5 are required for this repression. Specifically, the repression of Runx2 by Smad3/HDAC4/5 confers TGF-[unreadable] inhibitory signals to osteocalcin and Runx2 expression, and to osteoblast differentiation in vitro. TGF-[unreadable] repression of Runx2 function appears to be operative and important in vivo as well. The appearance of a cleidocranial dysplasia (CCD) phenotype in both Runx2 mice and mice over-expressing TGF-[unreadable] in bone suggests that these molecules function in the same pathway to regulate craniofacial bone development in vivo, a hypothesis that will be tested in Aim 1. By crossing Runx2 mice with mice possessing mutations in TGF-[unreadable] or HDAC function, we will also determine the ability of these pathways to modulate the severity of CCD. The extent to which TGF-[unreadable] regulates Runx2-target gene expression through HDACs 4 and 5 in vivo will also be tested in Aim 1 using transgenic mice with specific defects in this signaling pathway. Understanding the molecular control of normal osteoblast differentiation in vivo will provide new insights into how this differentiation pathway can be manipulated. As TGF-[unreadable] and Runx2 regulate the expression of many components of the bone matrix, the intersection of these pathways may affect the mechanical quality of bone matrix. Preliminary data suggest that partial reduction of TGF-[unreadable] signaling increases several functionally relevant properties of bone, including intrinsic mechanical properties, mineral concentration, cortical thickness, trabecular bone volume, and resistance to fracture. These conclusions were derived by the novel combined application of atomic force microscopy (AFM) with nanoindenting capability, micro-Raman spectroscopy, X-ray tomographic microscopy (XTM), and 3-point bending to study bone in transgenic mice. In Aim 2, these methods will be used to determine if genetic disruption of the downstream targets of TGF-[unreadable], HDACS and Runx2, can also affect these bone properties. In Aim 3, the hypothesis that TGF-[unreadable] regulation of bone matrix mechanical properties and composition is Runx2 and HDAC4/5-dependent will be tested directly by evaluating the bone quality of crossed mice (i.e. Runx2 X HDAC5-/-) generated in Aim1. These studies may lead to identification of targets that can be therapeutically manipulated to promote formation of high-quality bone to repair developmental or post-surgical defects in craniofacial bone. [unreadable] [unreadable] [unreadable]